Total Hexosaminidase Research Articles

Thermodynamic study of β- N-acetylhexosaminidase enzyme heterogeneity in human seminal plasma

Background It has been suggested that the activity of β- N-acetylhexosaminidase (Hex) in seminal plasma may be used as a biochemical marker of azoospermia. The purpose of our study was to evaluate this hypothesis using a thermodynamic procedure developed to determine total Hex activity and that of its isoenzymes in this biological fluid. Methods Using the substrate 3,3′-dichlorophenolsulphoftaleinil N-acetyl-β- d-glucosaminide, a highly significant difference ( p<0.001) is found between the activation energy of Hex A (41.5 kJ/mol) and of Hex B (72.3 kJ/mol), making it possible to determine the activity of these isoenzymes from the apparent activation energy of the total Hex in seminal plasma. Results A significant difference between the normozoospermic and azoospermic groups was only found for Hex A isoenzyme activity ( p<0.05), although with considerable overlapping between the values of both groups. Significant partial correlations were found for the total Hex, Hex A and Hex B activities with the immobile spermatozoa count ( p<0.01) and for total Hex and Hex B with the dead spermatoza count ( p<0.05). In turn, Hex A had a significant partial correlation with the live spermatozoa count ( p<0.05); however, Hex activity in seminal plasma of acromosomal origin appears to be of little importance in quantitative terms. Conclusions It was not possible to confirm that total Hex activity in seminal plasma, or even of its isoenzymes Hex A and Hex B, is a suitable biochemical marker of azoospermia (efficiency≤67%). The thermodynamic procedure described may be a useful alternative for the study of the Hex enzyme heterogeneity in spermatozoa.

Does Serum Hexosaminidase Activity Play a Role in the Diagnosis of Strangulated Bowel Obstruction? An Experimental Study

Strangulation is associated with an increased risk of mortality and morbidity in patients with mechanical bowel obstruction. The accurate and early recognition of the presence of strangulation is important to allow safe nonoperative treatment. A number of studies have shown that there was no single and reliable test to detect or exclude the presence of strangulation. The aim of this study was to evaluate the role of serum hexosaminidase (Hex) levels in recognition of strangulation in an experimental model of closed loop small bowel obstruction. Forty-two Wistar albino rats were divided into four groups: I, control (n = 5); II, sham laparotomy (n = 5); III, simple obstruction (n = 16); and IV, strangulation groups (n = 16). Activity levels of total Hex and its fractions (Hex A and B) were assayed in serum samples obtained from rats after 3 and 8 hr. Samples of small bowel were also evaluated histologically. Histological evaluation of bowel sections obtained from the strangulation group after 8 hr, revealed transmural hemorrhagic infarction in all animals with a mean +/- SD total Hex activity of 978.25 +/- 150 nmol/hr/ml, which was significantly higher than that in the other groups (P < 0.001). Although sections of bowel from the strangulation group after 3 hr showed severe ischemic injury, the activities of total Hex, Hex A, and Hex B were not different from those of the control, sham, and simple obstruction groups. Histological examination of these groups did not show any sign of ischemia. Total Hex, Hex A, and Hex B activities in the strangulation group were all significantly greater than the activities seen in the simple obstruction group (P < 0.001, for all). In conclusion, increased serum hex levels indicate irreversible transmural infarction only in the late period of strangulation in the closed loop small bowel obstruction model. It seems unuseful for detecting reversible and/or irreversible ischemia in the early period of strangulation.

Towards quality assurance in the determination of lysosomal enzymes: a two-centre study.

The definitive diagnosis of lysosomal storage disorders depends on the determination of enzymatic activities in cells, tissues or body fluids. At present, neither an evaluation of the different methods nor an interlaboratory quality assurance scheme is available. We have therefore determined the activities of total hexosaminidase, hexosaminidase A and beta-galactosidase in the same samples (n = 15) at two metabolic centres in Germany. Three different enzymatic methods were employed, two of which were based on leukocytes as enzyme source and one on dried blood spots. The results obtained by the two different methods using leukocytes proved comparable. In contrast, assays with dried blood spots showed poor correlation with results from leukocytes, possibly because enzymatic activity in dried blood is mainly derived from soluble plasma proteins. Nevertheless, accurate detection of a true enzyme deficiency was also possible in dried blood spots. All enzymes were highly stable when mailed frozen (recovery 98-120%). Enzymatic activities in dried blood samples were also stable at room temperature and were not affected even by exposure to elevated temperatures (50 degrees C for 3 h). Dried blood seems to be especially well suited for mailing from distant healthcare facilities, although more accurate results can be expected from leukocytes. In summary, comparability and pitfalls within a lysosomal quality assurance programme were evaluated.

Plasma and peripheral leukocyte β-N-acetylhexosaminidase isoenzymes and disease activity in rheumatoid arthritis

Objectives: Recently it has been suggested that serum β-N-acetylhexosaminidase (Hex) could be a joint destruction marker in rheumatoid arthritis (RA) patients. However, a large amount of serum Hex activity has its source from platelets, and the blood platelet-count is often increased in RA, which may have masked the significance of the results. The purpose of this study was to investigate the relationship between plasma activity of Hex and disease activity or severity. Design and Methods: In 51 patients with RA, with an evolution period for the illness of 10.9 ± 1.2 yr (range 1-40 yr), we determined the total Hex activity together with its Hex A and B isoenzymes in plasma and in mononuclear (MN) and polymorphonuclear (PMN) leukocytes. Results: The plasma activity of total Hex and Hex B isoenzyme was slightly higher in the group of patients studied ( p < 0.01), together with the specific activity of total Hex, Hex A and B in PMN leukocytes ( p < 0.001) than in the control group. No significant correlation was found between plasma or leukocyte Hex and the radiologic evaluation of the disease (Sharp’s modified method), or the patient’s functional capacity (modified Health Assessment Questionnaire). Likewise, a significant correlation between Hex activity and laboratory inflammation markers (C reactive protein, sialic acid, erythrocyte sedimentation rate) or the evolution time of the disease was not found. Conclusions: The plasma activity of total Hex, or even of its isoenzymes Hex A and Hex B, does not appear to be a reliable marker of erosion and cartilage degradation in RA patients. Liver function appears to be the major determinant for the plasma Hex activity in these patients.

Tay-Sachs and Sandhoff diseases: enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards

Background: Tay-Sachs disease (TSD), Sandhoff disease (SD) and variants are caused by deficient activity of the lysosomal enzymes hexosaminidase A (HA) and total hexosaminidase (TH) (hexosaminidase A plus B), respectively. For diagnosis, these enzymes are usually measured in plasma or extracts of leukocytes. We describe methods for the assay of hexosaminidase A and total hexosaminidase activities in dried blood spots (DBSs) on filter paper. Materials and Methods: We studied 163 healthy controls, 9 Tay-Sachs patients, 4 Sandhoff patients, 18 obligate carriers and the newborn-screening cards from two patients with Tay-Sachs and one patient with Sandhoff disease. To tubes containing a 3-mm-diameter blood spot, we added elution liquid and substrate solution. After incubation at 37 °C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. Results and conclusions: The described methodology is useful to distinguish patients with Tay-Sachs disease or Sandhoff disease from carriers and controls using samples that are sufficiently stable to be transported to the testing laboratory by mail. The diagnosis of both diseases from a newborn-screening card (NSC) was clearly demonstrated, even after storage for up to 38 months at room temperature. The newborn-screening card has been added to the biological materials that allow the identification of patients with Tay-Sachs disease and Sandhoff disease.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes.

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ss-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ss-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ss-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ss-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ss-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

Relationship between plasma ammonia concentration and beta-N-acetylhexosaminidase isoenzyme activities in liver cirrhosis.

Ammonia is known to increase the secretion of beta-N-acetylhexosaminidase (Hex) to the extracellular medium in cultured human fibroblasts and Hep G2 cells. We examined 35 patients with liver cirrhosis and the results revealed a significant increase for the plasma activities of total Hex and its isoenzymes Hex A and Hex B (p < 0.001). The partial correlations, with other biochemical markers of liver injury constant, between plasma ammonia concentration and the activity (r = 0.658) and the proportion in percentage of the Hex B isoenzyme (r = 0.692) were statistically significant (p < 0.001). The increased concentration of ammonia could explain, at least partially, an increased secretion of Hex B isoforms to the plasma in patients with liver disease.

Fenotipo infantil tardío y juvenil de la variante B1 de gangliosidosis GM2

Variant B1 is a rare form of GM2-gangliosidosis characterized by the presence of a mutation in the hexosaminidase A gene (HEXA) leading to a defect in the catalytic region of the alpha-subunit of beta-hexosaminidase A (alpha beta heterodymer). The mutated Hex A has almost normal activity against the natural synthetic substrates (4-methylumbelliferyl-N-acetyl-beta-D-glucosamine, 4MU-NAG) but is unable to hydrolyse GM2-ganglioside and the sulphated synthetic substrates (4MU-NAGS). The first and more frequent mutation described in the alpha-subunit gene associated to B1 variant GM2-gangliosidosis was a G533-->A transition (DN allele) resulting in Arg178His substitution. Here, we report the clinical, enzymatic and molecular characterization in two variant B1 late infantile and juvenile cases. Both cases presented regression of mental skills leading to dementia, epilepsy and severe motor impairment with dystonic involuntary movements and quadriplegia. In the late infantile case (death at 5 years and 8 months), cherry-red spot was also present. Enzymatic assays were performed in fibroblasts, leukocytes and serum and confirmed the abnormally low beta-hexosaminidase A activity against sulphated substrate despite a normal or nearly normal total hexosaminidase activity (unsulphated substrates). The patient with the late infantile phenotype was found to be compound heterozygote for the DN allele whilst the juvenile form was homozygote for that mutation. Variant B1 form of GM2-gangliosidosis is a rare and heterogeneous condition that must be kept in mind when evaluating neurodegenerative disorders associated with speech or gait disturbances, dystonia, seizures and pyramidal features.

A frequent TG deletion near the polyadenylation signal of the human HEXB gene: occurrence of an irregular DNA structure and conserved nucleotide sequence motif in the 3' untranslated region.

While screening for new mutations in the HEXB gene, which encodes the beta-subunit of beta-hexosaminidase, a TG deletion (deltaTG) was found in the 3' untranslated region (3'UTR) of the gene, 7 bp upstream from the polyadenylation signal. Examination of DNA samples of 145 unrelated Argentinean individuals from different racial backgrounds showed that the deltaTG allele was present with a frequency of approximately 0.1, compared with the wild-type (WT) allele. The deletion was not associated with infantile or variant forms of Sandhoff disease when present in combination with a deleterious allele. Total Hex and Hex B enzymatic activities measured in individuals heterozygous for deltaTG and a null allele, IVS-2 + 1G-->A (G-->A), were approximately 30% lower than the activities of G-->A/WT individuals. Analysis of the HEXB mRNA from leukocytes of deltaTG/WT individuals by RT-PCR of the 3'UTR showed that the deltaTG allele is present at lower level than the WT allele. By polyacrylamide gel electrophoresis, it was determined that a PCR fragment containing the +TG version of the 3'UTR of the HEXB gene had an irregular structure. On inspection of genes containing a TG dinucleotide upstream from the polyadenylation signal we found that this dinucleotide was part of a conserved sequence (TGTTTT) immersed in a A/T-rich region. This sequence arrangement was present in more than 40% analyzed eukaryotic mRNAs, including in the human, mouse and cat HEXB genes. The significance of the TG deletion in reference to Sandhoff disease as well as the possible functional role of the consensus sequence and the DNA structure of the 3'UTR are considered.

Diarrhea and autonomic dysfunction in a patient with hexosaminidase B deficiency (Sandhoff disease)

The causal factors and the physiopathology of motor diarrhea are still unclear. This case report describes a 60-year-old white man with severe diarrhea for more than 10 years and minor signs of autonomic dysfunction. Extensive investigation showed that small intestinal motility and absorption were normal but that accelerated colon transit precluded water and solute absorption from the large bowel. Orthostatic hypotension, sexual dysfunction, and loss of sweating suggested dysfunction of the autonomous nervous system, which was confirmed by reduced plasma concentrations of norepinephrine and dopamine. Rectal biopsy specimens showed enlarged enteric ganglion cells filled with lipidic material. Levels of total hexosaminidase and hexosaminidase B in plasma, white blood cells, and fibroblasts were decreased, as found in Sandhoff disease. The pedigree of the proband's family showed several affected and heterozygous individuals, detected by examination of total hexosaminidase and hexosaminidase B levels in plasma. Among the five homozygous subjects, three had a clinical picture of diarrhea and Orthostatic hypotension since the age of 50. Therefore, hexosaminidase B deficiency should probably be regarded as a cause for dysautonomia; dysfunction of the gastrointestinal tract, manifested by motor diarrhea or esophageal dysmotility, could be the initial and prevalent presentation of dysautonomia.

Detection of Tay-Sachs disease carriers among individuals with thermolabile hexosaminidase B.

The determination of hexosaminidases A and B in most programmes for Tay-Sachs disease carrier detection is based on their different heat sensitivity (hexosaminidase A is the heat labile isoenzyme). This routine cannot be employed for individuals who also possess a thermolabile hexosaminidase B. In Israel, 0.6% of the screened samples have a labile hexosaminidases B (about 110 each year) and the assessment of their hexosaminidase A activity has hitherto been based on isoenzyme separation by ion exchange chromatography. The latter requires relative large serum samples, and the individuals must usually be reappointed. In order to avoid the thermal treatment we have used the alternative technique, which employs two substrates with different specificities for the two isoenzymes: 1. The fluorogenic substance, 4-methylumbelliferyl-N-acetyl-glucopyranoside, which measures total hexosaminidase activity and 2. the derivative, 4-methylumbelliferyl-N-acetyl glucosamine-6-sulphate, which is considerably more specific toward hexosaminidase A. Hexosaminidase A activity was expressed as a ratio of total activities (the ratio of the assay with 4-methylumbelliferyl-N-acetyl glucosamine-6-sulphate to that with 4-methyllumbelliferyl-N-acetyl-glucopyranoside). Using the results from 65 obligate heterozygotes for Tay-Sachs disease, we established our reference ranges for assigning the genotypes with respect to the Tay-Sachs gene. Comparison of the results from 182 unrelated and randomly chosen sera screened by the ratio method and by heat inactivation, showed a very high correlation (r = 0.996). Sixty eight sera with thermolabile hexosaminidase B were tested by ion exchange chromatography and by the double substrate method, and they yielded identical diagnoses with regard to the Tay-Sachs locus.(ABSTRACT TRUNCATED AT 250 WORDS)

Degranulation of individual mast cells in response to Ca2+ and guanine nucleotides: an all-or-none event.

Widespread experience indicates that application of suboptimal concentrations of stimulating ligands (secretagogues) to secretory cells elicits submaximal extents of secretion. Similarly, for permeabilized secretory cells, the extent of secretion is related to the concentration of applied intracellular effectors. We investigated the relationship between the extent of secretion from mast cells (assessed as the release of hexosaminidase) and the degranulation (exocytosis) responses of individual cells. For permeabilized mast cells stimulated by the effector combination Ca2+ plus GTP-gamma-S and for intact cells stimulated by the Ca2+ ionophore ionomycin, we found that exocytosis has the characteristics of an all-or-none process at the level of the individual cells. With a suboptimal stimulus, the population comprised only totally degranulated cells and fully replete cells. In contrast, a suboptimal concentration of compound 48/80 applied to intact cells induced a partial degree of degranulation. This was determined by observing the morphological changes accompanying degranulation by light and electron microscopy and also as a reduction in the intensity of light scattered at 90 degrees, indicative of a change in the cell-refractive index. These results may be explained by the existence of a threshold sensitivity to the combined effectors that is set at the level of individual cells and not at the granule level. We used flow cytometry to establish the relationship between the extent of degranulation in individual rat peritoneal mast cells and the extent of secretion in the population (measured as the percentage release of total hexosaminidase). For comparison, secretion was also elicited by applying the Ca2+ ionophore ionomycin or compound 48/80 to intact cells. For permeabilized cells and also for intact cells stimulated with the ionophore, levels of stimulation that generate partial secretion gave rise to bimodal frequency distributions of 90 degrees light scatter. In contrast, a partial stimulus to secretion by compound 48/80 resulted in a single population of partially degranulated cells, the degree of degranulation varying across the cell population. The difference between the all-or-none responses of the permeabilized or ionophore-treated cells and the graded responses of cells activated by compound 48/80 is likely to stem from differences in the effective calcium stimulus. Whereas cell stimulated with receptor-directed agonists can undergo transient and localized Ca2+ changes, a homogeneous and persistent stimulus is sensed at every potential exocytotic site in the permeabilized cells.

Release of β-hexosaminidase isoenzymes in cultured human fibroblasts

We have used enzyme immunoassay methods to study the intra- and extracellular isoenzyme pattern of β-hexosaminidase (Hex) in human fibroblast cultures. The released activity into the medium during 24 h was about 10% of the intracellular activity. Intra- and extracellular ratio of Hex B to total Hex (Hex A plus Hex B) was about one-third. Estimation of the molecular weight of the released activity showed that it corresponded to 150 kDa, which is equal to the high molecular mass precursor forms of the enzyme. NH 4Cl is known to disturb the intracellular transport of lysosomal enzymes and increase the secretion of newly synthesized precursor forms. Addition of NH 4Cl even at 3 mM resulted in an increased release of total Hex, which was already noted within 24 h. We speculate that the increased concentration of plasma NH 4 + in patients with liver disease interferes with the intracellular distribution pathway of the lysosomal enzymes and this could contribute to the increased content of lysosomal enzymes present in plasma from these patients. Tunicamycin (0.3–3.0 μg/l) increased the percentage of Hex B in the medium, whereas an increased release of Hex was noted only after 48 h. Tunicamycin is known to enhance the secretion of N-linked oligosaccharide-free forms of lysosomal enzymes. Thus the oligosaccharide chains on α- and β-subunits seem to be important for the normal formation process of the Hex A isoenzyme.

Serum β‐Hexosaminidase Isoenzyme: A Sensitive Marker for Alcohol Abuse

Serum beta-hexosaminidase (Hex) isoenzymes analyzed by enzyme immunoassay were investigated in a group of alcoholics (n = 38) hospitalized for detoxication, in another group of alcoholics (n = 22) abstinent between 6 days and 10 years and in a reference group (n = 20). Hex "B" isoenzyme was elevated in all 38 patients hospitalized for detoxication but only 35 of these had total Hex values above the upper limit of the reference group. The Hex A isoenzyme, gamma-glutamyltransferase, and aspartate aminotransferase showed considerable overlap between these patients and the reference group. In the group of 22 abstinent patients only one had an increased level of Hex A, Hex "B," and total Hex, whereas 10 had gamma-glutamyltransferase values above the upper limit of the reference group. It is concluded that Hex "B" is a useful marker for alcohol abuse.

Immunoassay of β-hexosaminidase isoenzymes in serum in patients with raised total activities

Enzyme immunoassay (EIA) methods with monoclonal antibodies specific for β-hexosaminidase (Hex) isoenzymes A and B in human serum are presented. The proportion of Hex A obtained with the EIA-methods was similar to that found with ion-exchange chromatography and isoelectric focusing (about 60% of total Hex activity). The EIA-method for Hex B reacted to a similar extent with Hex B, Hex P and intermediate forms. A highly significant correlation was obtained between total Hex activity in human sera assayed with a conventional enzyme substrate method and the total Hex activity obtained as the sum of Hex A and Hex B analyzed with the EIA-methods. In sera from individuals with increased total activity of Hex there was a positive correlation between total activity of Hex, and percent of Hex B found with the EIA-methods. Investigation of sera from pregnant women and sera from patients with liver cirrhosis and cholestasis showed that the increase in total activity in these patient groups was mainly due to an increase of the isoenzyme forms reacting with the EIA-method for Hex B.

Adult onset motor neuronopathy in the juvenile type of hexosaminidase A and B deficiency

Two sisters presented with progressive muscle cramps, as well as wasting and weakness of the legs with onset after age 20. They also showed intention tremor of the upper extremities and dysarthria starting during the first decade. The older patient also had fasciculations; the younger, hyperreflexia. Total plasma beta-hexosaminidase (Hex) activity with 4-methylumbelliferyl-acetyl-glucosamine as substrate was reduced to 1.4% and 2.7% of the control in the 2 patients, respectively. Hex A activity measured by 4-methylumbelliferyl- N-acetylglucosamine-6- O-sulphate as substrate was 9.9% and 12.8% of the mean control value in the 2 patients, respectively. Hex B activity was undetectable in both patients. Leukocyte total Hex activity was 7–8% of normal; residual Hex A activity in the 2 patients was 17.8% and 16.3% of normal controls, respectively. Fibroblastic residual Hex A activity in the 2 patients was 9.6% and 22% of normal mean value, respectively. Appendiceal ganglion cells contained membranous cytoplasmic bodies in the younger patient. Thin layer chromatography of the appendiceal extract from one patient ( III 2 ) showed a marked increase of GM 2 ganglioside, and some increase of GM 3 ganglioside. Northern blots performed on fibroblast cell lines from both patients for the demonstration of alpha and beta locus messenger RNA showed no difference between patients and control. These patients have a rare form of adult-onset progressive motor neuron disease presumably due to abnormal beta subunits, causing severe deficiency of both Hex A and Hex B. The phenotypic expression of this disease is similar to motor neuron disease due to alpha locus mutations, which suggests that the Hex A deficiency, even though only a partial one, may be the important pathogenic factor.

Acute effects of oleic acid-induced lung injury in baboons

Oleic acid induces lung injury in the baboon. Within 90 min of intravenous oleic acid injection, there are significant increases relative to baseline in the bronchoalveolar lavage fluid (BALF) contents of total protein, lactic dehydrogenase, hexosaminidase, acid protease, and angiotensin-converting enzyme, indicating increased alveolar permeability. When BALF is chromatographed on Sephadex G-200, the elution profiles of hexosaminidase, acid protease, and angiotensin-converting enzyme in BALF are similar to those from normal plasma. SDS-polyacrylamide gel electrophoresis of BALF proteins indicates a similar molecular weight distribution in baseline and post-oleic-acid BALF. No evidence of serine proteases active in the inflammatory process, and present in many cases of human adult respiratory failure, is found either by incubating BALF with hemoglobin and elastin substrates or by labeling BALF with3H-diisopropylfluorophosphate. However, there is a significant increase in α1-antiprotease activity in BALF relative to the baseline value. The activities in plasma of several acute phase reactants including antiproteases, fibronectin, and angiotensin-converting enzyme do not change in this acute phase of the lung injury. The data indicate that many biochemical markers used to indicate acute respiratory failure in humans do not change acutely in the expected fashion in this animal model of the disease.

Serum Hexosaminidase as a Possible Biomarker for Human Cancer

The levels of total serum hexosaminidase and of its heat stable isozyme (beta-hexosaminidase) were determined in the sera of subjects bearing ovarian and other female genital cancers and of control subjects. The assays were carried out on a double blind basis. Total hexosaminidase levels were significantly elevated in sera of subjects with female genital cancers and in subjects with ovarian cancer. Levels of beta-hexosaminidase were elevated in all subjects with genital cancer. The hexosaminidase assay may be a convenient biomarker for human cancer since earlier studies have shown that total and beta-hexosaminidase levels are elevated in sera of most cancer patients studied.

Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoff's disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model.

312 DEVELOPMENT OF SERUM HEXOSAMINIDASE (HEX) IN INFANTS

Serum activity of hexosaminidases A and B has been measured to identify patients with Tay-Sachs and Sandhoff's diseases, and may also be affected in other clinical conditions including neonatal necrotizing enterocolitis (NEC). Data utilizing serum HEX as a marker for NEC is difficult to interpret because normal developmental patterns have not been characterized. We have measured serum activity of total HEX and Hex B in 61 neonates of 27 to 40 weeks gestation who did not have NEC. These infants were followed from birth until 4 to 8 weeks of age. The infants were divided into 2 gestational age groups (≤34 weeks and >35 weeks) since enzyme activities were similar within each of these 2 groups. Total serum HEX activity, increased with postnatal (PN) age and was greater in the older gestational age group (ANOVA p<0.01). Factors associated with increased total serum HEX include enteral feeding, parenteral nutrition and unexplained hematochezia. Serum HEX activity in infants with clinical conditions that may influence serum activity of this enzyme must be compared to that in normal infants of similar gestational and postnatal ages.